Wnt signaling is one of the most common pathways linked to carcinogenesis in the intestine. The transcription factors that mediate Wnt signals are members of the Lymphoid Enhancer Factor/T Cell Factor (LEF/TCF) family. This four-member family is complex in that there are full-length activating forms, truncated dominant negative forms, and alternatively spliced isoforms that modify Wnt target gene recognition. TCF-1 and TCF-4 are the two family members expressed in the epithelial crypt compartment of the intestine. Normally, TCF-1 is expressed as a dominant negative isoform and it counteracts the action of TCF-4 which is expressed as a Wnt-mediating, active isoform. This pattern changes in cancer because TCF-1 expression switches to a full-length, Wnt mediating form. We have discovered a second DNA binding domain in TCF-1 and TCF-4 isoforms called the C-clamp. We have shown that the C-clamp is important for regulating cell proliferation of colon cancer cells. We have also identified Wnts that trigger TCF-1 export in colon cancer cells. Export of TCF-1 leaves the C-clamp form of TCF-4 in the nucleus to mediate aberrant Wnt signaling. Three aims are proposed for this project. First, the extracellular Wnts and intracellular components that direct TCF-1 nuclear export will be defined (Aim1). Second, the biological impact of export will be defined in colon cancer cells and colon cancer initiating 3D cultures (Aim2). Third, the DNA binding specificities and target gene selection of C-clamp forms of TCF-1 and TCF-4 will be defined through genome-wide binding assays (ChIP-Seq), high-throughput protein binding microarrays, and in vitro biochemical analysis (Aim3). The overall goal is to define the mechanisms behind changes in TCF expression in colon carcinogenesis, and to define how extracellular Wnts and DNA binding specificities of TCFs influence aberrant signaling in colon cancer cells with stabilized -catenin.